Means and method for chemical analysis

ABSTRACT

A RIGID OR SEMI-RIGID DISPOSABLE REACTION CONTAINER IS DISCLOSED FOR USE IN MEASURING THE EXTENT OR RATE OF OPTICAL DESNITY CHANGE PRODUCED BY A REACTION, WHICH CONTAINER IS PROVIDED WITH CHAMBER MEANS THAT PREFERABLY COMPRISES A WINDOWED CHAMBER AND AN AUXILIARY CHAMBER HAVING OPENINGS COMMUNICATING WITH THE INTERIOR THEREOF AND INTERCONNECTED BY A PASSAGE OF SMALL CROSSSECTION THROUGH WHICH SOLUTION FROM THE AUXILIARY CHAMBER CONTAINING A DISSOLVED KEY REACTANT MAY BE INJECTED INTO THE REACTION CHAMBER TO INITIATE THE CHEMICAL REACTION. THERE IS FURTHER DISCLOSURE OF A DISPOSABLE REACTION CONTAINER THAT IS IN THE FORM OF A CHAMBERED SLIDE WHEREIN THE OPENING OR OPENINGS PROVIDING ACCESS TO THE CHAMBER MEANS PREFERABLY OCCURS IN THE MARGIN OF THE SLIDE. THERE ALSO IS DISCLOSED A DISPOSABLE REACTION CONTAINER IN THE FORM OF SEPARABLE SECTIONS WHICH ARE ADAPTED TO HAVE COMPONENTS OF THE REACTION MIXTURE DEPOSITED THEREON IN INDEPENDTENT SEPARATED RELATION ON PREDETERMINED SURFACE AREAS, THE COMPONENTS BEING DEPOSITED IN SOLID FORM PREFERABLY BY LYOPHILIZATION BEFORE BRINGING THE SECTIONS OF THE CONTAINER TOGETHER IN FACE-TO-FACE ABUTTING SECURED RELATION AND MEANS BEING PROVIDED CONDUCIVE TO KEEPING THE DEPOSITED COMPONENTS SEPARATE FROM EACH OTHER IN THE COMMON CHAMBER. THE DISCLOSURE ALSO DESCRIBES CERTAIN PROCEDURES INCLUDING THE LOADING OF THE SECTIONS OF THE REACTION CONTAINER WITH THE SEPARATED QUANTITIES OF SOLID COMPONENTS OF THE RECTION MIXTURE FOLLOWED BY UNITING OF THE SECITONS IN FACE-TO-FACE RELATION AND THEREAFTER, WHEN MAKING AN ANALYSIS, INTRODUCING A SUITABLE SOLVENT AND ANY OTHER COMPONENT OR COMPONENTS OF THE REACTION MIXTURE, THE REACTION BEING INITATED BY THE INJECTION OF A KEY COMPONENT. FOR PURPOSES OF INCUBATION, THE DISPOSABLE REACTION CONTAINER PREFERABLY COMPRISES AN INCUBATION CHAMBER WHICH IS USED IN CONTROLLING TEMPERATURE AS BY PLACING THEREIN A METAL WHICH IS RESPONSIVE TO A THERMOSTATICALLY CONTROLLED FIELD FOR THE GENERATION OF HEAT.

Sept. 12, 1972 G. H. BROWN ETAI- 3,591,017

MEANS AND METHOD FOR CHEMICAL ANALYSIS Filed May 1970 2 Sheets-Sheet lUvl so P 'E L mkskyk-x INVENTO GEORGE H. B OW ROBERT J, EWING BY W WA0.21%

Sept. 12, 1972 G. H. BROWN ETAL 3,691,017

MEANS AND METHOD FOR CHEMICAL ANALYSIS Filed May 1970 2 Sheets-Sheet zFIG.3 FIG.4

INVENTOR GEORGE H- BR WN ROBERT J. EWING BY A RNEYS United States PatentO 3,691,017 MEANS AND METHOD FOR CHEMICAL ANALYSIS George H. Brown,Briclrtown, and Robert J. Ewing, Colts Neck, N.J., assignors toWorthington Biochemical Corporation, Freehold, NJ.

Filed May 6, 1970, Ser. No. 34,926 Int. Cl. C12k 1/04 US. Cl. 195-103.5R 25 Claims ABSTRACT OF THE DISCLOSURE A rigid or semi-rigid disposablereaction container is disclosed for use in measuring the extent or rateof optical density change produced by a reaction, which container isprovided with chamber means that preferably comprises a windowed chamberand an auxiliary chamber having openings communicating with the interiorthereof and interconnected by a passage of small crosssection throughwhich solution from the auxiliary chamber containing a dissolved keyreactant may be injected into the reaction chamber to initiate thechemical reaction. There is further disclosure of a disposable reactioncontainer that is in the form of a chambered slide wherein the openingor openings providing access to the chamber means preferably occurs inthe margin of the slide. There also is disclosed a disposable reactioncontainer in the form of separable sections which are adapted to havecomponents of the reaction mixture deposited thereon in independentseparated relation on predetermined surface areas, the components beingdeposited in solid form preferably by lyophilization before bringing thesections of the container together in face-to-face abutting securedrelation and means being provided conducive to keeping the depositedcomponents separate from each other in the common chamber. Thedisclosure also describes certain procedures including the loading ofthe sections of the reaction container with the separated quantities ofsolid components of the reaction mixture followed by uniting of thesections in face-to-face relation and thereafter, when making ananalysis, introducing a suitable solvent and any other component orcomponents of the reaction mixture, the reaction being initiated by theinjection of a key component. For purposes of incubation, the disposablereaction container preferably comprises an incubation chamber which isused in controlling temperature as by placing therein a metal which isresponsive to a thermostatically controlled field for the generation ofheat.

THE FIELD OF THE INVENTION This invention relates to method and meansfor chemical analysis and relates more especially to such method andmeans which lends itself to automated or semiautomated analysisprocedures. The invention is especially suited for use in connectionwith the analysis of body fluids such as blood, urine, and the like.

BACKGROUND OF THE INVENTION Especially in the field of biochemistry,there is a very extensive need for chemical testing from the point ofview of both quantitative and qualitative analysis per-'- phasicscreening, diagnostic testing and patient monitoring, it becomesapparent that the existing testing facilities are heavily taxed both asregards equipment and personnel. There have been a number of proposalsfor automatic or semi-automatic testing equipment and procedures.However, proposals heretofore made have had inherent shortcomings suchas complexity and cost, less than desired accuracy, limited flexibilityand the necessity for the employment of trained technical personnel.

OBJECTS OF THE INVENTION A principal object of this invention is toprovide simple and inexpensive means for performing analyses of thecharacter aforesaid with a high degree of accuracy and without thenecessity for employing highly trained technical personnel.

It is a further object of this invention to provide a disposablereaction container which is simple, made of inexpensive material anddevoid of moving parts that lends itself to automated handling in themeasurement of optical density relative to the transmission of lightthrough a predetermined thickness of the reaction mass in the disposablecontainer.

A further object of this invention is to provide a dis posable reactioncontainer that also serves as a vehicle for the separated storage ofreactants in solid form in common chamber means.

It is a further object of this invention to provide a disposablereaction container which is constructed so as to enable a chemicalreaction to be initiated by the introduction of a key component at adesired time.

It is a further object of this invention to provide a disposablereaction container which comprises means whereby after all components ofthe reaction mass have been placed in the reaction container thereaction mass may be brought to desired temperature and maintained atsaid temperature during a period of incubation.

THE GENERAL NATURE OF THE INVENTION This invention involves theemployment of a disposable reaction container which is composed of rigidor semirigid material and which comprises transparent windows inpredetermined spaced relationship to each other for the successivetransmission of light therethrough and through a layer of predeterminedthickness of the reaction mass within the container. When the referenceis to transparent windows the reference is general to the transmissionof light whether or not in the visible range. For example, for analysessuch as those principally contemplated herein it is common practice toemploy a spectrophotometer as a source of monochromatic light in thewave length range from 330 m to 600 m One wave length which is commonlyused is in ultraviolet light at a wave length of essentially 340 m Thedisposable reaction container comprises chamber means which not only isprovided with the transparent windows but also is provided with areasthat are adapted for the deposition thereon of reaction components insolid form and that in the finished commercial product does have solublereactants in solid form deposited thereon in out-of-contact relation.These areas preferably have ribs between them which protrude inwardlyinto the reaction chamber but do not extend across the width thereof,thereby facilitating the deposit of the solid reaction components in theseparated state so as to minimize the possibility of their deteriorationcaused by any premature commingling of the components. Moreover, each ofthe areas preferably is provided with a multiplicity of protrusionswhich assist in maintaining proper disposition of the solids within thechamber means of the reaction container and minimize flow on saidsurface when the components are initially applied in the form of asolution. In this way it is possible to provide as a commercial producta disposable reaction container having stored therein the variouscomponents of the reaction mass other than the solvent and other thanthe sample which is to be introduced in making the analysis.

It is a further feature of this invention that the reaction container isinitially made in separate sections so as to facilitate the depositionof the solid components at the desired areas within the chamber means ofthe reaction container. In typical practice of the invention solutionscontaining predetermined quantities of dissolved solid reactioncomponents are deposited on selected areas of the sections and thereaction components are reduced to solid form by lyophilization. Afterthis has been accomplished the sections are united in assembledrelation. In normal construction each section comprises one of thetransparent windows and the sections are brought into face-to-facerelationship. Any suitable uniting means may be employed for holdingthem in position. Preferably the uniting means that is employedcomprises relatively movable surfaces in frictional contactingrelationship so that the sections may be secured together merely by theapplication of pressure. The employment of tongue and grooveinterfitting parts is well suited for accomplishing a press fit and frictional holding of the sections together. An adhesive may be usedadditionally or alternatively but ordinarily an adhesive is unnecessary.

A further feature of the invention resides in the employment of achambered slide comprising interior chamber means provided with anopening or openings communicating with the interior thereof. Preferablythe opening or openings are located at the margin of the slide. Thisconstruction coupled with the employment of rigid or semi-rigid materialfor the reaction container enables the reaction container to be handledas a relatively thin slide or plate which can 'be held with the openingor openings upwardly. This configuration and construction of thedisposable container enables the container to be readily handled byautomated mechanisms when carrying out an analysis or test in the mannerthat will be described more in detail hereinbelow.

It is a further feature of this invention that the chamber means that isemployed comprises a reaction chamber in combination with an auxiliarychamber. The reaction chamber has an opening therein communicating withthe interior thereof through which the solvent and the sample may beintroduced. The auxiliary chamber also has an opening communicating withthe interior thereof that is upwardly disposed when the reactioncontainer is in position with the opening for the reaction chamberupwardly disposed. The reaction chamber and the auxiliary chamber are incommunication with each other through a passage of reduced cross-sectionwhich ordinarily is disposed in portions of the reaction chamber and theauxiliary chamber that are remote from the aforesaid openings. When thereaction container is in the form of a chambered slide the openings forthe reaction chamber and for the auxiliary chamber preferably aredisposed in adjacent relation at one of the margins of the slide. Whenemploying chamber means comprising both a reaction chamber and anauxiliary chamber reaction components may be deposited on certain areasof the interior surface of the reaction chamber and another anddifferent reaction component specimen to be tested are provided in solidform in the reaction chamber and in the auxiliary chamber as hereinabovedescribed. When it is desired to make an analysis a solvent such aswater is introduced into the reaction chamber in a predetermined amountin relation to the capacity of the reaction chamber and of the auxiliarychamber such that the windows in the reaction chamber are covered. Thesample thereafter is added and since the solvent previously has flowedinto the auxiliary chamber very little or none of the component in theauxiliary chamber upon becoming dissolved migrates into the reactionchamber. When the contents are in this condition, the contents of thereaction container may be brought to desired temperature for thereaction and also may be permitted to incubate for such period of timeas may be desired. When, however, it is desired to initiate thereaction, the solution of the key component in the auxiliary chamber isforced therefrom through the restricted passage into the reactionchamber and upon the injection of the solution of the key component intothe reaction chamber from the auxiliary chamber it becomes commingledwith the solution of the other components in the reaction chamber,including the sample, and the reaction is initiated. The ribs in thereaction chamber and the protrusions hereinabove mentioned assist insetting up turbulence during the injection. In order to insure thoroughand complete commingling of the reactants, the solution in the mixingchamber may be caused to flow back into the auxiliary chamber whereuponthe injection may be repeated several times.

The forceful ejection of the solution in the auxiliary chamber throughthe passage of reduced cross-section, preferably is accomplished by theemployment of a puff of compressed air. To this end, the opening intothe auxiliary chamber may be round for the reception of a nozzle havinga conical exterior surface at the end of a line which supplies the puffof compressed air. The return of solution into the auxiliary chamber maybe accomplished by the pressure exerted by gravity. However, suchpressure may be assisted either by subjecting the interior of theauxiliary chamber to a short interval of subatomospheric pressureexerted through the opening and nozzle of an air line into the openinginto the reaction chamber, which opening in such case ordinarily wouldbe round for the reception of a nozzle having an exterior cone-shapedsurface. The insertion of the nozzle in either opening may beaccomplished manually. However, this invention contemplates the handlingof the chambered slides by equipment such that at the appropriate timethe nozzle or nozzles are brought into operative relation with theopening or openings in the slide followed by their disengagement whenthe desired mixing has been accomplished.

It is a further feature of preferred practice of this invention toinclude in the disposable reaction container, incubation meanscomprising a chamber which serves the purpose of bringing the reactionmixture to a desired temperature during the period of incubation beforeinitiating the reaction. In preferred practice of the invention theincubation chamber may be one which is disposed in contiguous relationwith the other chamber means and it may contain a metal distributedtherethrough which when heated assists in bringing the reactioncontainer and its contents to desired temperature and in maintainingdesired temperature. The metal within the container may be heated byconduction, from a continuation of the metal extending to the exteriorthat is heated as by contact with a heated holder or air or in responseto energy received from an energizing field such as a high frequencyelectric field.

In carrying out the method of the invention, the disposable reactionchamber is initially made up so as to contain solid components of thereaction mixture preferably deposited by lyophilization in situ on thesurfaces of the sections of the container before they are united witheach other. Because of the construction and configuration of the surfaceareas of the chamber means a number of diiferent reaction components maybe deposited in the container in solid form and in predeterminedquantities appropriate for the contemplated analysis. As will beillustrated more concretely hereinafter, the chamber means of achambered slide may readily present as many as eight or more individualsurfaces on which components may be deposited in solid form. Theparticular components that are employed in any case depends, of course,on the nature of the analysis that is to be performed. It is one of theadvantages of this invention that it has great flexibility in itscapacity to accommodate to an automatic or semi-automatic procedure awide variety of analyses and tests notwithstanding very greatdifferences in the nature and number of reaction components.

When it is desired to use the reaction container in the form wherein thechamber means includes an auxiliary chamber, all that is required is toadd a predetermined amount of water and the sample, e.g. a smallquantity of blood serum. The container may desirably be disposed inautomatic equipment with the openings disposed upwardly for thereception of the solvent and the sample. After introduction of thesolvent and the sample, the container which has now been charged withall of the ingredients of the reaction mass, ordinarily is brought to apredetermined temperature for carrying out the reaction. And when anincubation period is required, the desired temperature is maintainedduring the incubation period. After initiation of the chemical reactionby forceful injection of solution containing a key component from saidauxiliary chamber into said reaction chamber, the reaction container isbrought into operative relation with a beam of monochromatic light froma suitable source such as a spectrophotometer, and readings may be madewhich reflect optical density according to known procedures, thereaction container itself serving in effect as a cuvett'e. When the rateof reaction is to be determined, then optical density readings may betaken at stated intervals from which the rate of change in opticaldensity may be computed.

While this invention lends itself to the employment of any combinationof reactants and components appropriate for use in the reaction mass inthe performance of tests and analyses involving reactions that have aneffect on optical density, including more especially those carried outin an aqueous medium using water as the solvent, it is one of theadvantageous features of this invention that it is well suited forcarrying out reactions of the enzymatic type involving enzymaticreagents. Any of the reaction components normally used in such reactionsmay be employed such as enzymes, co-enzymes, selected substrates andsuch other components as may be desired such as buffers and inorganicsalts.

When carrying out the invention utilizing a reaction container whereinthe chamber means comprises a reaction chamber in combination with anauxiliary chamber containing the key component, and utilizing enzymaticreagents comprising a substrate, it is normally preferred that thesubstrate be initially deposited within the auxiliary chamber inasmuchas it is preferable to employ the substrate as the key component whichinitiates the reaction upon a solution thereof being injected into thereaction chamber.

While it is preferable to employ a reaction container having a windowedreaction chamber in combination with a contiguous auxiliary chamber thatis separate from the reaction chamber except for a passage of reducedcrosssection, it is within the purview of some of the aspects of thisinvention to employ only a single chamber, namely, the reaction chamber.In such case, all of the reaction components may be deposited in solidform on areas within the reaction chamber with the exception of the keycomponent, which in this case ordinarily is the sample. When theanalysis is to be performed then the solvent is introduced into thechamber and the contents of the reaction container may then be broughtto desired temperature during an incubation period, as has beendescribed hereinabove. When it is desired to initiate or trigger thereaction, a solution of the sample is introduced into the reactionchamber and mixed with the contents thereof as by the employment of ajet of air or the introduction of a stirrer. Thereafter, the extent ofany effect on optical density may be observed as described hereinabove.

DETAILED DESCRIPTION OF THE INVENTION Further objects, features andadvantages of this invention will become apparent from the followingdescription in connection with a specific embodiment of the disposablereaction container as illustrated in the accompanying drawings, wherein:

FIG. 1 is a perspective view of a disposable reaction container in thepreferred form of a chambered slide;

FIG. 2 is a cross-section on an enlarged scale of the chambered slidealong the line 2-2 of FIG. 1;

FIGS. 3 and 4 are, respectively, plan views of the separated sections ofthe chambered slide in the direction of lines 33 and 44, respectively,as shown in FIG. 2; FIGS. 3 and 4 being on a somewhat smaller scale ascompared with FIG. 2;

FIG. 5 is a schematic elevation of the slide section corresponding withFIG. 3 illustrating the liquid level that is assumed in the reaction andauxiliary chambers;

FIG. 6 is similar to FIG. 5 and illustrates schematically in connectionwith the complementary section of FIG. 4 the forcing of liquid from theauxiliary chamber into the reaction chamber responsive to a puif of airwith resultant mixing in the reaction chamber;

FIG. 7 shows the chambered slide in perspective in relation to a sourceof air under pressure and to a source of light emanating from a source;and

FIG. 8 corresponds with FIG. 4 but on a smaller scale and isillustrative of an alternative chamber arrangement.

Having reference to the illustrative embodiment shown in the drawings,the disposable reaction container is shown in its preferred form,namely, in the form of a chambered slide, which is indicated generallyby the reference character 10. The slide is composed of two abuttingplanar sections 11 and 12 which are shown in FIG. 2 as being held inface-to-face relation by tongues 13 which inter fit with grooves 14 insliding frictional relation such that the sections may be forcedtogether by pressure and detachably held together by the frictionalcontact between the interfitting surfaces of the tongues and grooves.The arrangement of parts in section 11 is shown in FIG. 3 and thearrangement of parts in section '12 is shown in FIG. 4. As is apparentfrom the drawings, the arrangements are complementary so that thedifferent portions of each section oppose each other when the sectionsare brought together as shown in FIG. 2. 'In the central portion ofsection 11 there is a transparent window 15 which is in opposed spacedrelation with respect to the window '16 in section 12.

In the central portion of the slide planar extended chamber means isprovided. In the embodiment shown the larger portion of the chambermeans in the region of the windows is the reaction chamber \17. Thechamber means also comprises the auxiliary chamber 18 which is separatedfrom the reaction chamber 17 by a wall 19 except for a passageway 20 ofreduced cross-section. The reaction chamber is provided with an opening21 which communicates with the interior thereof and the auxiliarychamber 18 is provided with an opening 22 that communicates with itsinterior. It is to be noted that in the preferred embodiment shown inthe drawings, the openings 2-1 and 22 are located in proximity to eachother in one of the margins of the chambered slide and that when theslide is in position with one of the openings disposed upwardly, theother opening also is disposed upwardly. The passage is shown asdisposed according to preferred practice in the portion of the chambermeans opposite to the openings 21 and 22.

The inner surface of the section 11 that is in the vicinity of thewindow 15 is provided with a multiplicity of small protrusions therefromwhich, as shown, are in the form of a succession of small ridges 23.Similarly, the portion of section 12 of the slide adjacent the window 16has small ridges 24 protruding therefrom. In section 11 the ribs 25separate the inner surface of the chamber into three areas which areindicated by the reference characters 26, 27 and 28, Similar ribs 29border the window 15'. In section -12 the ribs 39 and 40 subdivide thearea surrounding the window 16 into areas 30, 31 and 32. The ribs 25,29, 39 and 40 protrude into the reaction chamber but do not extendthereacross.

As regards the auxiliary chamber 18, the inner surface likewise presentsprotrusions 33 in the form of small ridges.

When the sections 11 and 12 are secured in face-to-face relationship,the tongue and groove jointure about the chamber means provides means toretain a liquid therein when the openings 21 and 22 are disposedupwardly.

In the preferred embodiment shown, there is an additional chamber 35which borders the central chamber means which holds the reaction mass.The chamber 35 is referred to herein and in the claims as an incubationchamber. It provides means that is part of the reaction container itselfwhereby the reaction mass after its completion, except for thecommingling of the key component with the other components, may bebrought to desired temperature and maintained at desired temperatureduring an incubation period. In the embodiment shown the chamber 35 hasmetal disposed therein which conveniently may be aluminum foil 38.Aluminum is a metal which is a good conductor of heat and which whensubjected to an energized field such as high frequency electricity,absorbs energy in the form of heat. These properties may be utilized inbringing the slide and its contents to desired temperature and inmaintaining desired temperature. In the embodiment shown a continuation38A of the aluminum foil within the chamber is extended so as to overliea substantial portion of the external surface of the slide other thanthe windows. When utilizing the slide, it is contemplated that aplurality of the slides will be disposed in a suitable holder which maybe made of aluminum and which by its disposition in an environmentmaintained at the desired temperature becomes heated to substantiallythe same temperature. In such case, the aluminum foil overlying theexternal surface of the slide receives heat from the environment andlikewise as the result of contact with surfaces of the holder. The heatso received becomes transmitted by conduction to the foil within thechamber so that the container and its contents may be brought to desiredtemperature more quickly. Alternatively, the aluminum foil may be heatedby subjecting it to a high frequency electric field. In such case,automatic control may be provided by thermostatic control whichenergizes and de-energises the energizing field according to whether thetemperature goes below or above the predetermined temperature to bemaintained. The external continuation 38A of the foil may be employed tofacilitate detection of the temperature of the aluminum foil.Temperature control also may be had in other ways. Thus the chamber 35may be omitted and the desired temperature during incubation may beobtained by partial immersion of the slide in a body of liquidmaintained at a desired temperature. Alternatively, if desired, themetal foil may be omitted from the chamber 35 and by the employment ofsuitable inlet means and outlet means (not shown) for the chamber 35 afluid at a desired predetermined temperature may be flowed through thechamber 35 during the incubation period.

Typical usage of the chambered slide is illustrated in connection withthe following examples:

Example 1 According to this example, the chambered slide of thisinvention as illustrated in the drawings is employed in an analysis forthe determination of the amount of lactic dehydrogenase in a sample ofblood serum. When a person become afilicted with the heart conditionknown as myocardial infarction the body releases certain enzymes intothe blood stream. One of the enzymes so released is lactic dehydrogenaseand the presence of a more than normal amount of lactic dehydrogenase ina sample of blood serum is symptomatic of a myocardial infarctioncondition. The reagents employed in an analysis for the detection oflactic dehydrogenase are lactic acid and diphosphopyridine nucleotide(DPN) and a phosphate buffer.

Having specific reference to the chambered slide shown in the drawings,the components of the reaction mixture other than the water and thesample are first deposited on the desired surface areas of the slidewhile the sections 11 and 12 are in the separated condition asillustrated in FIGS. 3 and 4. In the reaction under presentconsideration, the lactic acid is the substrate and is the key componentwhich initiates or triggers the reaction. The lactic acid is depositedfrom solution on a surface area presented in the auxiliary chamber 18,namely, the surface 33 of section 11 or the surface 34 of section 12, orboth of these surfaces. A solution of DPN may be deposited on area 26 ofsection 11 or area 30 of section 12, or both. Buttered solution may beapplied to sections 27 or 28 or both of section 11 or sections 31 and 33or both of section 12 or on all four of these areas. After the solutionshave been deposited on the respective areas above mentioned, thesolutions are reduced to solid form, preferably by lyophilization. Eachof the reagents is present in the predetermined amount for providing thedesired concentration thereof in the reaction mass when it is completedby the addition of water and the sample.

In typical practice of the invention, the chambered slide may be 2inches square and 7 inch in thickness, the distance between the innersurface of the windows being 5 mm. The dimensions of the windows are /2inch by inch. The diameter of the round opening communicating with theinterior of the reaction chamber is inch and the diameter of the roundopening communicating with the interior of the auxiliary chamber is 4;inch and the dimensions of the passageway 20 are A inch by 2.5 mm. Thecapacity of the chamber means is such that 1.4 ml. of water or othersolvent fills the chamber means to the level indicated in FIG. 5. Theslide may be made of any rigid or semi-rigid material such as a styreneor a clear acrylic resin subject to the windows having adequatetransparency for the monochromatic light used for the contemplated test.By rigid or semi-rigid, it is meant that the container structure havesufiicient rigidity so that the distance between the windows issubstantially accurately maintained at a predetermined spacing.

When the reaction components have been reduced to solid form, theyadhere to the different areas and remain separated from each other andwhen a metal such as aluminum foil is employed in the incubationchamber, it is put in place in one of the sections. The sections arethen brought to the position shown in FIG. 2, thereby completing theslide as a commercial product. The completed slide may be usedimmediately or packed in boxes for shipment and storage prior to use bya customer.

When the analysis is to be performed the operator adds 1.4 ml. of waterthrough the opening 21. During this step the added water flows into theauxiliary chamber. Thereafter 0.1 ml. of serum sample is introduced intothe reaction chamber. These additions may be accomplished in anysuitable way, as by the employment of a micropipette or a microsyringe.However, as regards the water addition, the

correct quantity of water can be measured into the slide when the amountrequired to fill to an indicated level is known. After the addition ofthe water and the serum sample, the slide and its contents are broughtto a constant temperature which, for the illustrative test in question,is from 30 to 37 C., depending on the setting that may be desired inthis range. For a given test the temperature should be brought within102 C. of the reaction temperature that is desired. This ordinarilyrequires about one minute, and to accomplish desired incubation thereaction mass may be maintained at this temperature for an additional 4minutes. When it is desired to initiate the reaction the opening 22 intothe auxiliary chamber is brought into operative relation with the nozzle36 of an air line 37 and the reaction is initiated by introducing intothe auxiliary chamber a small puff of air such as .5 cc. which forcesthe contents of the auxiliary chamber through the restricted passage 20into the reaction chamber 17 with a swirling action, as illustrated inFIG. 6. When this operation is performed manually a short length ofresilient rubber hose may be used that has a conical nozzle at the endthereof that fits into the opening 22 of the auxiliary chamber and theputt of air may be produced by squeezing the rubber hose. The mouth ofthe opening 22 may be somewhat flared so as to conform to the conicalsurface of the nozzle 36. The presence of the ribs protruding from thesurfaces of the reaction chamber assist in the mixing action. Ifdesired, air pressure in the auxiliary chamber. Alternatively, amechanical plunger could be restored, whereupon a second puif of air maybe employed to further the mixing action in the mixing chamber. Ifdesired, this may be repeated and more generally the flow back and forththrough the restricted passage 20 may be efiected by subatmospheric orsuperatmospheric pressure applied to either the auxiliary chamber or tothe reaction chamber. Alternatively, a mechanical punger could beemployed instead of air.

When the mixing of the reaction mixture has been completed, any lacticdehydrogenase in the blood sample acts as a catalyst which catalyzes thereaction to form as reaction products pyruvic acid and DPN in thereduced form (DPNH). Since the D'PNH, which is produced as a reactionproduct, has a substantially higher optical density for light at 340 muthan does DPN, the rate of any increase in optical density is a functionof the amount of lactic dehydrogenase in the sample of blood serum.After the initiation of the reaction as the result of commingling thesubstrate with the other reaction components, the reaction rate may bedetermined by moving the slide into position for the passage of light at340 m through the transparent windows of the slide and through thethickness of the reaction mass between the two windows which, for thepurposes of this example, may be 5 mm. Any change in optical density perunit of time may be measured and in known fashion the data thus obtainedmay be translated into values indicative of the quantity of lacticdehydro genase contained in the sample of blood serum. In an analysissuch as that exemplified, ten readings at intervals of one minute areadequate.

Example 2 The practice of this invention may be further illustrated byits employment in the determination of creatine phosphokinase (*CPK).The presence of CPK in blood serum is generally indicative of musculardisease. The reagents employed are creatine, adenosine triphosphate,glutathione, phosphoenol pyruvate, pyruvate kinase, lactatedehydrogenase, reduced diphosphopyridine nucleotide (DPNH) and aphosphate buifer.

In preparing the slide the creatine, which is a substrate and functionsas the key component, is caused to be deposited in solid form on thesurface area within the auxliary chamber. The other components may bedeposited in any convenient Way on surface areas that are presented inthe reaction chamber. If desired, certain of the compatible reactioncomponents may be combined such as the glutathione with the phosphatebuffer. The two enzymes may likewise be combined. When the analysis isto be carried out, water is added followed by a sample of blood serum,and the slide and its contents are brought to desired reactiontemperature and are subjected to incubation, as above described inconnection with Example 1, followed by initiation of the reaction by theinjection of the solution of creatine into the reaction chamber and themaking of optical density readings at stated intervals, as alsodescribed in connection with Example 1. When the reaction mixture iscompleted the reaction of creatine and adenosine triphosphate iscatalyzed by any creatine phosphokinase in the blood serum with theproduction of creatine phosphate and adenosine diphosphate. Pyruvatekinase in turn catalyzes the reaction of adenosine diphosphate withphosphoenol pyruvate with reformation of adenosine triphosphate andpyruvate which is thus made available for the indicator reaction whereinthe lactate dehydrogenase catalyzes the reaction between pyruvate andthe reduced diphosphopyridine nucleotide (DPNH) with resultant formationof lactate and diphosphopyridine nucleotide (DPN) which has less opticaldensity for monochromatic light-at 340 m than DPNH. In this analysissuccessively timed determinations of decreasing optical density areindicative of the rate of the reaction and in turn the concentration ofcreatine phosphokinase in the sample of blood serum.

It is to be understood that the foregoing is solely for the purpose ofillustrating a typical embodiment and utilization of the invention. Thusthe reaction container may occur in other forms or in otherconfigurations within the purview of the invention as herein disclosedand claimed. One such modification is illustrated in FIG. 8 which isgenerally similar to FIG. 4 wherein the parts which are essentially thesame are indicated by like reference characters. The principalmodification which distinguishes FIG. 8 from FIG. 4 is that the Wall 19is provided with the extension 19A which as shown is disposed inparallelism with the bottom of the chamber means and is spaced therefromby the buffer zone 18B which is an extension of the primary zone 18A ofthe auxiliary chamber 18. The passage 20 of reduced cross-section isdisposed as indicated in FIG. 8, i.e., at a location that is separatedfrom primary zone 18A by the longitudinal extent of the buffer zone 18Bwhich ordinarily is of substantially greater longitudinal extent thanwidth. The auxiliary chamber 18, including the zone 18B, is shown aspresenting the ridged surface areas 39 in zone 18A and 40 in zone 18Bseparated by the rib 41. The reaction chamber 17 is shown as presentingthe ridged surface areas 42, 43 and 44 which are separated from eachother and from the window 16 by the ribs 45.

FIG. 8 shows one section only of the complete slide. The other sectionmay be the complement in all respects of that shown in FIG. 8. However,this is not necessarily the case. Thus, the arrangement of surface areasmay be different as compared with FIG. 8, or alternatively, the surfacespresented by the interior of the auxiliary and reaction chambers may beplain when it is not regarded as necessary to deposit any of thereaction components therein.

The chamber arrangement shown in FIG. 8 may be desired to provideadditional assurance that during the incubation period none of the keycomponent migrates from the auxiliary chamber into the reaction chamberas, for example, if a prolonged incubation period is desired or if thecomponents of the reaction mass are such as to be conducive to rapiddiffusion of the key component. When utilizing the embodiment shown inFIG. I the key component, such as the substrate of an enzymaticreaction, ordinarily is deposited on the ridged surface of the primaryzone 18A while one or more other and difierent reactants is or aredisposed on the ridged surface of any or all of the areas 42, 43 and 44presented by the interior surface of the reaction chamber. The ridgedsurface of the buffer zone 18B of the auxiliary chamber may have nothingdeposited thereon or may have deposited thereon some soluble componentof the reaction mass, such as a buffer, that is inert with respect tothe component deposited in zone 18A. In either case, when the reactioncontainer is put into use by the introduction of water into the reactionchamber the water flows into the auxiliary chamber via the opening 20and the zone 18B before entering zone 18A. Notwithstanding solution ofthe key component in zone 18A, the possibility during incubation ofmigration by diffusion of any of the key component from zone 18A intothe reaction chamber is doubly insured by the necessity for travelthrough the buffer zone 18B as well as through the passage 20 of reducedcrosssection. If the zone 18B is not utilized for the deposition thereinof a soluble solid component of the reaction mass, the buffer zone 18Bmay approach the cross-sectional dimensions of the passage 20 ofrestricted cross-section and thereby provide an elongated passage whichmay have substantially the same restricted cross-sectional dimensionsthroughout its length.

While the opposed sections of the reaction container have beenillustrated as utilizing tongue and groove members adapted tofrictionally hold the sections together and provide fluid-tightperipheral walls for the auxiliary and reaction chambers and provide afluid-tight wall between the auxiliary and reaction chambers, othermeans may be provided. Thus, if desired, prevention of accidentalseparation may be assured by the employment of conventional cooperatinglatch members that resiliently snap into locked position when theopposing sections are pressed together.

It is to be understood that the foregoing examples are merelyillustrative of the flexibility and adaptability of the invention todifferent tests and analyses and to automated diagnostic and analyticprocedures.

We claim:

1. A disposable reaction container which is composed essentially ofrigid or semi-rigid material and the walls of which present interiorsurfaces that define a reaction chamber, and

an auxiliary chamber, said container having a first opening therein thatis in communication with the interior of said reaction chamber,

a second opening that is in communication with the interior of saidauxiliary chamber and that is upwardly disposed when said container isin position with said first opening upwardly disposed,

said container having a passage of restricted cross-sectioncommunicating between said reaction chamber and said auxiliary chamberadjacent the portions thereof remote from said first and secondopenings, said passage being adapted and arranged for the injection ofliquid from said auxiliary chamber into said reaction chamber so as tobecome commingled with liquid in said reaction chamber upon forcingliquid from said auxiliary chamber through said passage,

said reaction chamber having transparent windows therein adapted andarranged for the transmission of light therethrough and through a bodyof predetermined thickness of liquid in said reaction chamber betweensaid windows, and

said auxiliary chamber and said second opening in communicationtherewith being adapted for being placed in operative relation with asource of gas under pressure and for imposing gas pressure supplied fromsaid source on liquid in said auxiliary chamber for forcing it into saidreaction chamber through said passage.

2. A disposable reaction container according to claim 1 wherein a firstsoluble reaction component in solid form is within said reaction chamberand a second soluble reac tion component in solid form that is differentfrom said first component is within said auxiliary chamber.

3. A disposable reaction container according to claim 2 wherein saidsecond component is a key reaction component.

4. A disposable reaction container according to claim 2 wherein saidsecond reaction component is a substrate for an enzymatically inducedreaction with said first reaction component.

5. A disposable reaction container which is composed essentially ofrigid or semi-rigid material and the walls of which present interiorsurfaces that define a reaction chamber, and

an auxiliary chamber, said container having a first opening therein thatis in communication with the interior of said reaction chamber,

a second opening that is in communication with the interior of saidauxiliary chamber and that is upwardly disposed when said container isin position with said first opening upwardly disposed,

said container having a passage of restricted cross-sectioncommunicating between said reaction chamber and said auxiliary chamberadjacent the portions thereof remote from said first and secondopenings, said passage being adapted and arranged for the injection ofliquid from said auxiliary chamber into said reaction chamber so as tobecome commingled with liquid in said reaction chamber upon forcingliquid from said auxiliary chamber through said passage,

said reaction chamber having transparent windows therein adapted andarranged for the transmission of light therethrough and through a bodyof predetermined thickness of liquid in said reaction chamber betweensaid windows, and

said container comprising as an integral part thereof incubating meanscomprising a chamber that is in contiguous thermally-conductive butnon-interconnected relation with respect to said reaction chamber andwith respect to said auxiliary chamber and that is adapted to contain asubstance heated to a desired incubating temperature whereby thecontents of said chambers may be heated to and maintained at saidincubating temperature.

6. A disposable reaction container according to claim 5 wherein saidincubating chamber contains metal adapted to be heated responsive to anenergy-imparting electric field.

7. A disposable reaction container according to claim 5 wherein saidincubating chamber contains a heat-conductive metal and wherein saidmetal has a continuation thereof of substantial extent disposed on theexterior of said reaction container.

8. A disposable reagent-containing reaction container in the form of achambered slide which is composed essentially of rigid or semi-rigidmaterial and which comprises interior planar extended chamber meanshaving an opening communicating with the interior thereof,

said slide comprising planar sections in abutting faceto-face relation,

a transparent window in each of said sections, said windows being onopposite sides of said chamber means and adapted and arranged for thesuccessive transmission of light therethrough and through a portion ofthe chamber means therebetween,

means securing said sections together in face-to-face fluid-tightrelation for the retention in said chamber means when said opening isdisposed upwardly of liquid introduced into said chamber means throughsaid opening, and

a surface area presented by at least one of said sections havingdeposited thereon in solid form a predetermined quantity of a solublereaction component.

9. A disposable reactant-containing reaction container according toclaim 8 wherein said soluble reaction component is deposited in solidform on said area by lyophilization in situ.

10. A disposable reactant-containing reaction container according toclaim 8 wherein the surface of said area having said reaction componentdeposited therein presents a multiplicity of protrusions which arerestrictive of flow of liquid along said surface.

11. A disposable reactant-containing reaction container according toclaim 8 wherein said chamber means presents a plurality of surface areashaving different reaction components in predetermined quantity depositedthereon.

12. A disposable reaction container in the form of a chambered slidewhich is composed essentially of rigid or semi-rigid material and whichcomprises an internal planar extended reaction chamber having an openingcommunicating with the interior thereof,

said slide presenting planar sections in abutting faceto-face relation,

a transparent window in each of said sections, said windows being onopposite sides of said reaction chamber and adapted and arranged for thesuccessive transmission of light through a portion of the reactionchamber therebetween,

means securing said sections together in said abutting face-to-facerelation and in fluid-tight relation for the retention in said chamberwhen said opening is disposed upwardly of liquid introduced into saidchamber through said opening, and

at least one of said sections presenting a plurality of surface areasadjoining each other and the window therein, adjoining areas beingseparated from each other by a rib that projects into and only partiallyacross the width of said reaction chamber.

13. A disposable reaction container in the form of a chambered slidewhich is composed essentially of rigid or semi-rigid material and whichcomprises a planar extended reaction chamber having an opening thereinaifording communication with the interior thereof,

incubating means comprising a chamber laterally disposed with respect tothe periphery of said reaction chamber in contiguousthermally-conductive noninterconnected relation therewith,

said slide presenting abutting planar sections in complementaryface-to-face abutting relations,

a transparent window in each of said sections, said windows being onopposite sides of said reaction chamber and adapted and arranged for thetransmission of light successively therethrough and through a portion ofthe reaction chamber therebetween,

means securing said sections in face-to-face abutting relation forretention in said reaction chamber when said opening is disposedupwardly of liquid introduced into said chamber through said opening,and

said chamber comprised in said incubating means being adapted to containa heated substance whereby the contents of said reaction chamber may beheated and maintained at a desired incubating temperature.

14. A disposable reaction container according to claim 13 wherein saidincubating chamber contains metal adapted to be heated.

15. A disposable reaction container in the form of a chambered slidecomposed essentially of rigid or semirigid material, said slidecomprising a planar extended reaction chamber,

a first opening in the margin of said slide in open communication withthe interior of said reaction chamber,

an auxiliary planar extended chamber laterally adjacent said reactionchamber and separated from said 14 reaction chamber except for a passageof reduced cross-section communicating between said chambers adjacentthe portions thereof remote from said margin of said slide having saidopening therein,

a second opening in said margin of said slide that is separated fromsaid first opening by a wall that separates said reaction chamber fromsaid auxiliary chamber except for said passage, said second openingbeing in proximate spaced relation with respect to said first openingand in open communication with the interior of said auxiliary chamber,and

a transparent laterally disposed window in each wall of said reactionchamber, each window being in spaced relation relative to the other andsaid windows being disposed for the successive transfer of lighttherethrough and through the portion of the reaction chambertherebetween.

said chambered slide comprising abutting planar sections incomplementary face-to-face relation, the inner surfaces of which definesaid reaction and auxiliary chambers and each of which has one of saidwindows therein, and

securing means which secures said sections together in face-to-faceliquid-tight relation for the retention of liquid therein when saidopenings in said slide are disposed upwardly.

16. A disposable reaction container according to claim 15 wherein saidsecuring means comprises opposing surfaces in frictional detachableengagement.

17. A disposable reaction container according to claim 15 wherein saidsecuring means comprises cooperating latching members.

18. A disposable reaction container in the form of a chambered slidecomposed essentially of rigid or semirigid material, said slidecomprising a planar extended reaction chamber,

a first opening in the margin of said slide in open communication withthe interior of said reaction chamber an auxiliary planar extendedchamber laterally adjacent said reaction chamber and separated from saidreaction chamber except for a passage of reduced cross-sectioncommunicating between said chambers adjacent the portions thereof remotefrom said margin of said slide having said opening therein,

a second opening in said margin of said slide in proximate spacedrelation with respect to said first opening and in open communicationwith the interior of said auxiliary chamber, and

a transparent laterally disposed window each wall of said reactionchamber, each window being in spaced relation relative to the other andsaid windows being disposed for the successive transfer of lighttherethrough and through the portion of the reaction chambertherebetween, and

at least one of said sections presenting a surface area on the interiorof said reaction chamber having a first reaction component depositedthereon in solid form and in predetermined amount and at least one ofsaid sections presenting an interior surface area of said auxiliarychamber having a second reaction component deposited thereon that isdifferent from said first reaction component and that is in solid 1 formand in predetermined amount.

19. A disposable reaction container according to claim 18 wherein saidsecond component is a substrate enzymatically reactive with said firstcomponent and wherein said first and second reaction components aredeposited on their respective surface areas by lyophilization in situ.

20. A disposable reaction container according to claim 15 wherein saidauxiliary chamber comprises a primary zone and a buffer zone, saidbuffer zone being interposed between said primary zone and said openingof reduced cross-section, effecting substantial separation of said pri-15 mary zone from said opening and having substantially greater lengththan width.

21. In a method for determining change in optical density resulting froma chemical reaction wherein the reaction is carried out in a reactioncontainer provided with spaced transparent windows arranged forsuccessive transfer of light therethrough and through a portion of thereaction mixture disposed therebetween and wherein components of thereaction mixture are dissolved in a solvent medium and the reaction isevidenced by the change in optical density following completion of thereaction mixture, the improvement which comprises placing at least oneof the components of the reaction mixture in solid form in said reactionchamber, placing a key component in solid form essential for initiatingthe reaction in an auxiliary chamber of said reaction container that isadjacent said reaction chamber and is separated from said reactionchamber except for a passageway of restricted cross-section, introducingthe solvent medium of the reaction mixture into said reaction chamberand thence into said auxiliary chamber in such manner that there is nosubstantial return flow of said solvent medium containing dissolved keycomponent from said auxiliary chamber into said reaction chamber,thereby effecting solution of said solid components in said auxiliarychamber and in said reaction chamber in said solvent medium, introducinginto said reaction chamber any other component required to complete thereaction mixture, and thereafter forcing solution containing said keycomponent in dissolved condition from said auxiliary chamber throughsaid passage into said reaction chamber thereby commingling it with thesolution in the reaction chamber and initiating the reaction.

22. A method according to claim 21 wherein after 16 the introduction ofsaid solvent medium the reaction container and its contents is broughtto a predetermined reaction temperature prior to forcing solution fromsaid auxiliary chamber into said reaction chamber.

23. A method according to claim 21 wherein solution of said keycomponent is forced from said auxiliary chamber into said reactionchamber by application of air pressure.

24. A method according to claim 21 wherein after introduction of thesolution of the key component into the reaction chamber a portion of theresulting mixture is returned to the auxiliary chamber and forcefulinjection into the reaction chamber is repeated.

25. A method according to claim 21 wherein said key component in asubstrate of an enzymatically catalyzed reaction with a component insaid reaction chamber and wherein after the solvent medium has beenintroduced a material comprising the catalyzing enzyme is introducedinto the reaction chamber prior to forceful injection of solution of thesubstrate into the reaction chamber.

References Cited UNITED STATES PATENTS 2,995,425 8/1961 Fuhrmann 23253 R3,233,975 2/1966 McCormick 103.5 R X 3,083,145 3/1963 Ryan 19554 X A.LOUIS MONACELL, Primary Examiner M. D. HENSLEY, Assistant Examiner US.Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,691,017 Dated September l2. 1972 Inventor(s) George H. Brown andRobert J. Ewing It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Col. 9, line 29, delete "chamber. Alternatively, a mechanical plungercould" and substitute --cha.mber may be relieved so that the liquidlevel may-;

line 36,-delete "punger" and substitute --plunger--.

Col. 13, line M9, delete "relations" and substitute -relation-.

Col. l t, line 17, delete the period (r) and substitute a comma line 50,after "window" insert -in-.

Signed and sealed this 13th day of February 1973..

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents FORM PO-105O (10-69) USCOMM-DC 60376-P69 U 5, GOVERNMENTPRINTING OFFICE 1 I965 0-365-334

